1. Field of the Invention
The present invention relates generally to the field of single nucleotide polymorphism genotyping. More specifically, the present invention provides a real-time polymerase chain reaction-based genotyping assay for the detection of single nucleotide polymorphisms.
2. Description of the Related Art
P-glycoprotein (P-gp), a member of the large adenosine triphosphate-binding (ATP-binding) cassette superfamily of transport proteins also called traffic ATPases, is the product of the human multidrug resistance gene (MDR1). P-glycoprotein is highly expressed on the apical (luminal) surface of organs that have excretory functions, such as the bile canalicular membrane of hepatocytes and the renal proximal tubule. Moreover, P-glycoprotein is significantly expressed on the luminal surface of tissues that serve as barriers, such as the brush border of the small intestine and the capillary endothelial cells of the blood-brain barrier.
Tissue distribution suggests that P-glycoprotein protects the body from toxic xenobiotics by secreting them into the bile, urine, and intestinal lumen and by reducing their accumulation in the brain and testes. As a result, interindividual variability in the disposition of numerous drugs has been ascribed to differences in P-gp expression. It has been reported that intestinal P-glycoprotein expression accounted for approximately 30% of interindividual variability in the maximal plasma concentration after oral administration of cyclosporine.
A novel P-glycoprotein aberrant allele, MDR1*2, linked to 2 synonymous single nucleotide polymorphisms (SNPs) (C1236T in exon 12 and C3435T in exon 26) and a nonsynonymous single nucleotide polymorphism in exon 21 (G2677T, Ala893Ser) was recently described (Kim et al., 2001). The single nucleotide polymorphisms found on exons 12, 21, and 26 are not strictly allelic; however, they exhibit strong linkage disequilibrium and account for a majority of the described haplotypes (Kim et al., 2001; Tang et al., 2002). MDR1*2 was found to be associated with altered fexofenadine disposition. Individuals carrying 2 wild type alleles (*1/*1) had a 40% greater fexofenadine systemic exposure after oral administration compared with individuals heterozygous or homozygous for MDR1*2. Reduced fexofenadine systemic exposure in carriers of the MDR1*2 allele potentially results in reduced therapeutic benefit after oral administration of fexofenadine.
Kim et al. (2001) reported significant ethnic differences in MDR1*2 allelic frequency, with 62% and 13% of European Americans and African Americans, respectively, carrying at least one MDR1*2 allele. Thus, polymorphic MDR1 expression may contribute to interracial variability in drug disposition. Unfortunately, attempts to determine the association between polymorphic P-glycoprotein expression and drug disposition have yielded equivocal results.
To facilitate clarification of the significance of commonly occurring MDR1 single nucleotide polymorphisms and their ethnic frequency on drug disposition, a rapid and robust polymerase chain reaction-based (PCR-based) screening method for the single nucleotide polymorphisms C3435T and G2677T would be highly desirable. The present invention fulfills this longstanding need and desire in the art.